This invention relates to novel phosphoepoxides, to the process for the production thereof and to the use thereof for stimulating Tγ9δ2 lymphocytes bearing TCR receptors with Vγ9 and Vδ2 variable regions.
In healthy individuals, the Tγδ lymphocytes of primates (humans, monkeys) present in the peripheral bloodstream usually constitute from 1 to 5% of the lymphocytes in the blood and play a role in the immune system. It has been demonstrated that they recognize their antigenic ligands by direct interaction with the antigen without presentation by molecules of the MHC by a presenting cell. Tγ9δ2 lymphocytes (sometimes also known as Tγ2δ2 lymphocytes) are Tγδ lymphocytes bearing TCR receptors with Vγ9 and Vδ2 variable regions. They constitute the majority of Tγδ lymphocytes in human blood.
When activated, Tγδ lymphocytes exercise a strong cytotoxic activity which is unrestrained by the MHC and is particularly effective in killing various types of cells, in particular pathogenic cells. These may be cells infected by viruses (“γδ T cell activation or anergy during infections: the role of nonpeptidic TCR ligands and HLA class I molecules” Fabrizio POCCIA et al, Journal of Leukocyte Biology, 62, 1997, p. 1–5), or by other intracellular parasites, such as mycobacteria (“The antituberculous Mycobacterium bovis BCG Vaccine is an attenuated Mycobacterial producer of phosphorylated nonpeptidic Antigens for human γδ T cells” Patricia CONSTANT et al, Infection and Immunity, vol. 63, no. 12, December 1995, p. 4628–4633); or by protozoans (“Plasmodium falciparum stimuli for human γδ T Cells are related to phosphorylated Antigens of mycobacteria” Charlotte BEHR et al, Infection and Immunity, Vol. 64, no. 8, 1996, p. 2892–2896). They may also be cancer cells (“CD94/NKG2 inhibitory receptor complex modulates both antiviral and antitumoral responses of polyclonal phosphoantigen-reactive Vγ9 Vδ2 T lymphocytes” Fabrizio POCCIA et al, Journal of Immunology, 159, p. 6009–6015; “Stimulation of γδ T cells by phosphoantigens” Jean-Jacques FOURNIE, Marc BONNEVILLE, Res. Immunol., 66th FORUM IN IMMUNOLOGY, 147, p. 338–347).
It has been demonstrated that, in the event of a mycobacterial infection, human Tγ9δ2 lymphocytes react to four natural, nonpeptidic molecules of a phosphorylated structure, known as phosphoantigens, which exhibit stimulation activity at a concentration of 1 to 5 nM (nanomolar) (WO-95/20673 and “Stimulation of human γ δ T cells by nonpeptidic Mycobacterial ligands” Patricia CONSTANT et al, Science, 264, p. 267–270).
These natural antigens have not been completely identified. Certain authors have erroneously presented them as alkene derivatives of pyrophosphate, in particular isopentenyl pyrophosphate IPP (U.S. Pat. No. 5,639,653 and “Natural and Synthetic nonpeptide antigens recognized by human γδ T cells”, Yoshimasa TANAKA et al, Nature, 375, 1995, p. 155–158). It has nonetheless now been demonstrated that none of these prenyl pyrophosphates is active at a concentration of nanomolar magnitude. The best results which have been obtained have been unable to demonstrate activity at below 3 μM for IPP and below 0.3 μM for dimethylallyl-UTP and 3-methyl-2-hexene pyrophosphate. The minimum active concentration of these compounds is thus, at best, of the order to 100 times higher than that of natural phosphoantigens.
With regard to IPP, it should in particular be noted that the most recent of the above-stated publications make the mistake of deducing the structure of the isopentenyl radical solely on the basis of mass spectrometry and the detection of a certain level of bioactivity. Indeed, apart from the fact that the compound analyzed in these publications was not purified and that a mass spectrum cannot identify uncharged species, it may be demonstrated that there are in fact several thousand different chemical structures which may have the same molecular weight and be a substituent of pyrophosphate in these molecules.
The fact that the minimum active concentration for IPP is much higher (some 1000 times higher) and that the intensity of the Tγ9δ2 lymphocyte responses obtained is much weaker than that for natural phosphoantigens demonstrates that IPP is not one of these natural phosphoantigens (“A novel nucleotide-containing antigen for human blood γδ T lymphocytes”, Y. Poquet et al, Eur. J. Immunol. 1996, 26, p. 2344–2349). This is moreover confirmed by numerous other observations: IPP is not found in sufficient concentration in mycobacterial extracts which stimulate Tγ9δ2 lymphocytes; according to “High pH anion exchange chromatographic analysis of phosphorylated compounds: application to isolation and characterization of non peptide mycobacterial antigens”, Y. Poquet et al, Anal. Biochem, 243 no. 1, 1996, p. 119–126, IPP does not have the same chromatographic (HPAEC) characteristics as natural phosphoantigens; IPP and other natural isoprenoids are produced by all living cells, but these do not stimulate Tγ9δ2 lymphocytes.
Moreover, it is known that substances having bioactivity of the order of or greater than 1 μM are only rarely compatible with the economic constraints of operation on an industrial scale. The synthetic phosphoantigens which have hitherto been proposed thus cannot be processed on an industrial scale under acceptable economic conditions.
Natural phosphoantigens, on the other hand, may only be produced in very small quantities (WO 95/20673) and, since their precise chemical structure has still not yet been established, they cannot be synthesized. Economic industrial scale processing is thus likewise out of the question, despite their demonstrated great therapeutic worth.